Page 29 - LC-SFC_Pharma_Brochure

2. System Volume and Gradient
3. Corr
ection of Gradient Delay
2. System V olume and Gradient 3. Correction of Gradient Delay
Delay
Delay
We have discussed differences in chromatograms caused by method
System volume differences must be considered when transferring a transfer and the origin of these differences. Next, we describe an ex-
method from one system to other systems. ample analysis and method transfer using multiple LC systems that
have different system volumes.
Fig. 2 shows the flow line from the mobile phase reservoir to the
column of LC system. Gradient delay volume means the system
volume between the point where two or more eluents are mixed and 3-1. Alkylphenone Analysis
3-1. Alkylphenone Analysis
the column inlet. As shown in Fig. 2, the gradient delay volumes are
different for low-pressure gradient and high-pressure gradient sys- Alkylphenones are aromatic ketones that can be analyzed by reversed
tems. Even for the same type of gradient system, different lengths phase chromatography. We analyzed alkylphenones using two systems
and/or internal diameters of piping can provide different gradient of different system volumes and compared the chromatograms ob-
delay volumes. tained from each system. We then adjusted the difference in retention
time between the two systems by adding an initial hold time.
Fig. 3 shows how gradient delay affects separation. In general, even
if gradient has already started on the time program, the actual gradi- The results are shown in Fig. 4. A comparison of the two chromato-
ent start time (time to increase an organic solvent concentration) is grams (Fig. 4, top and middle) shows differences between the peaks in
delayed. If the result obtained from a system with a large system the latter half of the chromatograms. These chromatograms were ob-
volume (Fig. 3, right) is compared to that from a system with a small tained from analyses performed under identical conditions but differ-
system volume (Fig. 3, left), we can see the gradient start time is de- ent systems. The system volumes of system 1 and 2 are 1155 µL, 505
layed more. This can cause different separation patterns on different µL, respectively. Therefore, there is a difference of 650 µL between the
systems. two systems. This difference in the system volumes leads to different
separation. The chromatogram at the bottom of Fig. 4 was obtained
Consequently, system volume difference must be considered when
transferring a method and the gradient program must be modified by via an analysis using system 2, after adding an initial hold time equiva-
making an adjustment to the initial hold time (gradient start time). lent to 650 µL. This chromatogram is almost identical to that obtained
Nevertheless, gradient programs cannot be modified when the ana- from system 1; this confirms that equivalent results can be obtained
lytical conditions are strictly defined by the testing regulations. from different systems by correcting for system volume difference.

Gradient delay volume of a low-pressure gradient system

Autosampler
Pump Mixer

Column

Gradient delay volume of a
high-pressure gradient system

Fig. 2 Gradient Delay Volumes (System Volumes)

System volume (small) System volume (large)
B.Conc (%) B.Conc (%)
The gradient begins early when system volume is small. The gradient is delayed when system volume is large.
100 100

50 50

0 0
0.0 1.0 2.0 3.0 4.0 min 0.0 1.0 2.0 3.0 4.0 min
Fig. 3 System Volume and Gradient Delay

2